Set Up Cell Lines For Functional Study Of Ankylosing Spondylitis' ERAP Variant Genes In Vitro

AS is a common kind of systemic autoimmune disease,the most common member of the SpA family,sharing verlapping clinical manifestations with other SpAs,including psoriatic SpA,IBD-associated arthritis,reactive arthritis.with a significant impact on patients' quality of life.Disease onset is typically in the young male and can be latent for a long time,leading to diablity for delayed diagnosis and treatment in spite of advanced radiography method.Thus,AS is considered to have a great impact on patient and bring heavy economic burden on their family and society.AS is a chronic inflammatory arthritis predominantly affecting the spine and sacroiliac joints.AS causes joint pain and stiffness that can result in joint fusion and spinal deformities.nflammation is a critical component of AS that precedes bone formation,but understanding of the inflammatory processes in AS is limited.Currently,there is no cure for AS and the available treatment modalities suppress pain and inflammation in a proportion of patients only.Objective: The pathogenesis of AS has remained elusive.The strongest genetic association of AS is with HLA-B27 discovered more than 40 years ago,but we are not clear how it contributes to the pathogenesis.Following the advent of genome-wide association studies,the number of known loci associated with AS is more and more.Endoplasmic reticulum aminopeptidase is second only to HLA-B27 in the strength of association.They not only have gene-gene interaction,but also can act in the same pathogenic pathway.So we set up cell lines for functional interaction of Ankylosing Spondylitis' ERAP variant with HLA-B27 in vitro.Method: The most common subtypes associated with AS is HLA-B27:04(Han Chinese)and HLA-B27:05(Caucasian).We chose HLA-B27:04/B27:05+ AS patients and healthy control,then transformed their B cells to B lymphoblastoid cell line.In order to illuminate the association of ERAP variant gene with their altered peptide trimming function,mRNA from AS patients and controls were extracted and reversly transcribed to cDNA,whose ERAP1 and ERAP2 were sequenced.One or both of them were knocked out by the most advanced gene editing technology,CRISPR/Cas9.The knock-out cell lines can be used to study the ERAP trimming function alteration of producing HLA-B27:04/B27:05 restricted peptides by transfecting wild type and mutared ERAP1/ERAP2 gene from sequencing mentioned above.To differenciate the trimming function,several kinds of specific T cells should be set-up,including HLA-B27:04/B27:05 restricted allo-antigen specific T cells.Thus,HLA-B27:04/B27:05 molecular should be cloned to set up C1R-B27:04/B27:05 cell lines for stimulating T cells.Results: We have transformed the healthy control's and AS patients' B cell to B-LCL,whose ERAP2 gene have been knocked out by CRISPR/Cas9.Their ERAP1 and ERAP2 mRNA were sequenced to find natural ERAP1 and ERAP2 sequence.The haplotype of 5 AS-associated SNPs in ERAP1 in AS patients have no difference with control.However,the mutation of ERAP2 in AS patients is more than control,especially potencial new transcripts by alternative splicing.We have cloned HLA-B27:04 gene to vector in order to set up C1R-B27:04,which is used to stimulate HLA-B27:04 restricted allo-antigen specific T cells.Conclusion: Our strategy to set up system for studying ERAP variant function in vitro have worked.When T cells specific to HLA-B27:04/27:05 restricted peptides are set up,we can compare the ERAP variant function.And we can get more information when ERAP1 is knocked out.